Schüler W, Kloiber K, Matt T, Bister K, Konrat R
Institute of Organic Chemistry and Institute of Biochemistry, University of Innsbruck, Austria.
Biochemistry. 2001 Aug 14;40(32):9596-604. doi: 10.1021/bi010509m.
The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.
通过使用从13C、15N标记的蛋白质样品中获得的更多数量的NOE约束，并应用最近开发的用于确定主链二面角ψ的三共振交叉相关弛豫实验，鹌鹑CRP2（LIM2）的溶液结构得到了显著改善。此外，通过氮-羰基多量子弛豫探测了与主链角φ和ψ均相关的15N（i）-1HN（i）偶极子和13CO（i）CSA张量的相对取向，并将其用作细化CRP2（LIM2）中金属配位位点局部几何结构的附加约束。分别通过质子检测的13C'（i-1）-15N（i）和15N（i）-1HN（i）多量子弛豫对位于CRP2（LIM2）折叠部分的残基的主链动力学进行了表征。我们表明，在毫秒到微秒时间尺度上具有主链各向同性化学位移交叉相关时间调制的区域与突变蛋白CRP2（LIM2）R122A中结构改变的残基相关（破坏CCHC锌指稳定侧链氢键），并且这些残基是连接两个锌结合位点的扩展氢键网络的一部分。这表明在两个锌结合亚结构域中存在长程集体运动。LIM结构域的构象可塑性可能与这个重要的蛋白质识别基序的功能相关性有关。
